基于高通量测序技术的冠心病患者肠道菌群多样性研究

Diversity of Gut Flora in Coronary Heart Disease Patients by High-Throughput Sequencing

为了分析冠心病人群肠道菌群结构和多样性特征,探究冠心病人群与健康人群肠道菌群结构差异。研究共采集50例冠心病患者和35例健康人粪便样本,提取粪便样本总DNA;根据细菌16SrDNA V3~V5区设计引物进行扩增,利用Illumina Miseq平台进行高通量测序;测序结果经过Reads拼接,OTUs（operational taxonomic units）聚类,物种注释,α多样分析,主成分分析,最终得到样品物种信息。在冠心病组和健康组中,拟杆菌门（Bacteroidetes）和厚壁菌门（Firmicutes）的丰度最高,其中拟杆菌门分别占到46.94%和61.96%,厚壁菌门分别占到41.51%和30.58%;丰度较高的是变形菌门（Proteobacteria）、梭杆菌门（Fusobacteria）、放线菌门（Actinobacteria）。此外,两组菌群结构差异性表现为：冠心病组的拟杆菌门明显低于对照组（P=0.015〈0.05）;而冠心病组的厚壁菌门显著高于对照组（P=0.009〈0.01）。α多样性分析结果显示,两组人群各样本在细菌种群多样性方面没有明显差异。OTUs比对结果表示,Veillonella、Veillonella dispar、Scardovia、Mogibacterium致病菌在冠心病组显著增高,而Bacteroidetes门下Parabacteroides、Bacteroides uniformis、Bacteroides caccae等有益菌则显著降低。主成份分析显示,综合PC1、PC2、PC3因素冠心病组和对照组样本被较好的区分开,表明健康组和冠心病组菌群结构存在显著差异。这些结果表明,冠心病患者与健康人群在肠道菌群多样性方面没有明显差异,但在菌群结构上存在较大差异;同时冠心病人肠道菌群致病菌数量多于健康人群,而拟杆菌类有益菌低于健康人群。

In order to analyze the characteristics and diversity of gut microbiota in patients with coronaryheart disease（CHD）,and explore the differences of gut microbiota structure between coronary heart disease patients and healthy human,we collected 50 cases of coronary heart disease patients’ and 35 cases of healthy people’s stool samples and extracted the samples to get total DNA.The primers were designed on bacterial 16 SrDNA V3~ V5 region sequences and Illumina Miseq platform was used for high-throughput sequencing.Sequencing results were treated by splicing Reads,OTUs（Operational Taxonomic Units）clustering,species annotation,αdiversity analysis,principal component analysis.In the end,we got the species’ information of all samples.The results indicated that Bacteroidesand Firmicutes were the most abundant phylum in the healthy controls and patients with coronary artery disease.Bacteroides accounted for 61.96% and 46.94%,and Firmicutes took 30.58% and 41.51% respectively.Then secondly high abundance phylum were Proteobacteria,Fusobacterium,Actinobacteria.On the phylum classification level,the Bacteroides phylum bacteria were significantly higher in healthy control group than patients with coronary heart disease（P=0.015〈0.05）;but Firmicutes in healthy controls group were significantly lower than in coronary heart disease patients group（P=0.009〈0.01）.Each index ofαdiversity analysis was not significantly different between the two groups,which mean all samples in the two groups had no differences on bacteria population diversity.The results of OTUs comparison showed that the pathogenic bacteria including Veillonella,Veillonella dispar,Scardovia,Mogibacteriumin coronary heart disease were significantly higher,while the beneficial bacteria covering Bacteroidetes,Parabacteroides,Bacteroides uniformis,Bacteroides caccae were dramatically lower in CHD group.Principal component analysis showed that the healthy group and CHD group had different microbial community structure.The study showed that there was no obvious difference about the diversity of the intestinal flora between coronary heart disease patients and healthy volunteers.But the community structure represented drastic discrepancy between two groups.Meanwhile,the number of intestinal pathogenic bacteria in coronary heart disease patients was higher than healthy people,but some benefit bacteria had lower abundance in CHD patients than healthy people.